Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol.

211

236

Two heat-stable protein inhibitors of protein phosphatase 2A (PP2A), tentatively designated I1PP2A and I2PP2A, have been purified to apparent homogeneity from extracts of bovine kidney. The purified preparations of I1PP2A exhibited an apparent M(r) approximately 30,000 and 250,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-300, respectively. In contrast, the purified preparations of I2PP2A exhibited an apparent M(r) approximately 20,000 and 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-200, respectively. The purified preparations of I1PP2A and I2PP2A inhibited PP2A with 32P-labeled myelin basic protein, 32P-labeled histone H1, 32P-labeled pyruvate dehydrogenase complex, 32P-labeled phosphorylase, and protamine kinase as substrates. By contrast, I1PP2A and I2PP2A exhibited little effect, if any, on the activity of PP2A with 32P-labeled casein, and did not prevent the autodephosphorylation of PP2A in incubations with the autophosphorylation-activated protein kinase [Guo, H., & Damuni, Z. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504]. The purified preparations of I1PP2A and I2PP2A had little effect, if any, on the activities of protein phosphatase 1, protein phosphatase 2B, protein phosphatase 2C, and pyruvate dehydrogenase phosphatase. With 32P-labeled MBP as a substrate, kinetic analysis according to Henderson showed that I1PP2A and I2PP2A were noncompetitive and displayed a Ki of about 30 and 25 nM, respectively. Following cleavage with Staphylococcus aureus V8 protease, I1PP2A and I2PP2A displayed distinct peptide patterns, indicating that these inhibitor proteins are the products of distinct genes. The N-terminal amino acid sequences of the purified preparations indicate that I1PP2A and I2PP2A are novel proteins.

Section: Resultsmentioning

confidence: 99%

Purification and Characterization of Two Potent Heat-Stable Protein Inhibitors of Protein Phosphatase 2A from Bovine Kidney

Li¹,

Guo²,

Damuni³

1995

211

236

“…Purification of Two Trimeric Forms of PP2A from Rabbit Skeletal Muscle. Two forms of PP2A were purified close to homogeneity from rabbit skeletal muscle by developing a rapid procedure which involved three or four chromatographic steps (Table 1) as compared with previously published protocols which required up to nine steps (Tung et al, 1985;Mumby et al, 1987;Waelkens et al, 1987;Usui et al, 1988). Purification involved one batch-adsorption on DEAE-cellulose, a DEAE-Sepharose-CL 6B, a poly(L-lysine)-agarose, and a thiophosphorylase a-Sepharose affinity chromatography.…”

Section: Resultsmentioning

confidence: 99%

“…We report here a rapid purification procedure which yielded only trimeric PP2A with no indication of any dimeric form. This result raises the possibility that, in previous preparations of two-subunit PP2A (Tung et al, 1985;Mumby et al, 1987;Waelkens et al, 1987;Usui et al, 1988;Chen et al, 1989), the B-subunit might simply have been lost from trimeric PP2A to generate the dimeric form. The B-subunit could have been dissociated from the trimeric PP2A or could have been subjected to degradation.…”

Section: Discussionmentioning

confidence: 98%

“…Another subunit with an apparent molecular mass of 54 kDa3 (the B' subunit) has been reported to be present in a form of phosphatase termed PP2A0 (Tung et al, 1985). However, isolation of this latter form of enzyme has since eluded several laboratories (Mumby et al, 1987;Waelkens et al, 1987;Usui et al, 1988;Chen etal., 1989). The bovine heart PP2A was originally described as containing a B subunit (Mumby et al, 1987), and therefore molecular weight marker glutamic dehydrogenase, whose deduced molecular mass is 55 kDa.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Diversity in the Regulatory B-Subunits of Protein Phosphatase 2A: Identification of a Novel Isoform Highly Expressed in Brain

Zołnierowicz¹,

Csortos²,

Bondor³

et al. 1994

115

101

The physiological role of type 2A protein phosphatases (PP2A) is dependent upon the association of the catalytic subunit with a variety of regulatory subunits. In order to understand the function of PP2A, we have undertaken purification of the holoenzymes and molecular cloning of the regulatory subunits. Two trimeric forms containing distinct B-subunits, PP2A0 and PP2A1, have been purified from rabbit skeletal muscle. The B-subunits associated with PP2A0 and PP2A1 migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with slightly different mobility, approximately 52.5 and approximately 51.5 kDa, respectively and showed distinct immunological properties. The B' form of B-subunit associated with PP2A0 was recognized by antibodies against the B-subunit present in bovine heart PP2A but not by antibodies specific to the B subunit isoforms of rabbit PP2A1. Cloning of cDNAs encoding the B subunit of PP2A1 resulted in the isolation of a cDNA highly homologous to, but distinct from, the B alpha subunit isoform. The deduced amino acid sequence of this novel isoform, which was designated B gamma, encoded a protein which was 81% and 87% identical to the B alpha and B beta isoforms, respectively. Northern blot analysis indicated that the B gamma isoform is highly expressed in rabbit brain as a transcript of 3.9 kb. Analysis of B-subunit expression by Western blot indicated a general parallel with the message levels. In conclusion, our data reveal even greater complexity of PP2A trimeric holoenzymes due to the identification of a novel B regulatory subunit isoform of PP2A1 and a distinct B' subunit associated with PP2A0.

“…In most tissues, PP2A is a heterotrimer composed of a 38-kDa catalytic subunit, a 65-kDa A regulatory subunit (RP65). and a B regulatory subunit with a molecular mass of 54-72 kDa or a heterodimer of the catalytic and 65-kDa regulatory subunit (Usui et al, 1988;Shenolikar & Nairn, 1991: Hendrix et al, 1993. Since the 65-kDa regulatory subunit is known to bind tightly to the catalytic subunit, we examined whether the NF-associated PP2A contained the 65-kDa regulatory subunit by immunoblotting.…”

Section: Nf-associatedmentioning

confidence: 99%

Neurofilament-associated protein phosphatase 2A: Its possible role in preserving neurofilaments in filamentous states

Saito¹,

Shima²,

Osawa³

et al. 1995

Neurofilament phosphatase (NF-phosphatase) activity, which dephosphorylates NF proteins phosphorylated by cyclic AMP-dependent protein kinase (A-kinase), was detected in NF fractions prepared from bovine spinal cords. This phosphatase was suggested to be associated with NFs by gel filtration and sedimentation analysis and was further demonstrated by dephosphorylation-dependent binding assay of NFs to microtubules. The NF-associated NF-phosphatase was identified as a type of protein phosphatase 2A (PP2A) by (i) its complete inhibition with 100 nM okadaic acid, at which concentration the purified type 1 protein phosphatase (PP1) was inhibited only 25%; (ii) the absence of effect of inhibitor-2, a specific inhibitor of PP1, on the NF-phosphatase activity; and (iii) the detection of 38-kDa catalytic and 65-kDa regulatory subunits of PP2A by immunoblotting. The NF-associated PP2A was partially solubilized from NFs by a high concentration of MgSO4, and the solubilized PP2A was suggested by gel filtration to be a dimeric holoenzyme consisting of a 38-kDa catalytic and a 65-kDa regulatory subunit. Phosphorylated NF-L, which is assembly incompetent, was induced to assemble into filaments by dephosphorylation with PP2A. These results suggest a role of NF-associated PP2A in preserving filamentous forms of NF in neurons.